U.S. patent application number 13/773749 was filed with the patent office on 2013-09-19 for micelle compositions and methods for their use.
This patent application is currently assigned to University of South Florida (A Florida Non-Profit Corporation). The applicant listed for this patent is Suraj Kishore Dixit, Mark Christian Howell, Shyam S. Mohapatra, Subhra Mohapatra, Chunyan Wang. Invention is credited to Suraj Kishore Dixit, Mark Christian Howell, Shyam S. Mohapatra, Subhra Mohapatra, Chunyan Wang.
Application Number | 20130243867 13/773749 |
Document ID | / |
Family ID | 49157871 |
Filed Date | 2013-09-19 |
United States Patent
Application |
20130243867 |
Kind Code |
A1 |
Mohapatra; Subhra ; et
al. |
September 19, 2013 |
MICELLE COMPOSITIONS AND METHODS FOR THEIR USE
Abstract
Provided herein is a micelle composition comprising a
polyethylene glycol (PEG), a DC-cholesterol, and a
dioleoylphosphatidyl-ethanolamine (DOPE) and either or both a
pharmaceutical compound core and a polynucleotide coating. Also
provided herein is a method of administering one or more compounds
to a cell comprising administering to the cell a micelle
composition comprising 1) PEG-PE, a DC-cholesterol, and DOPE, and
2) the one or more compounds, wherein the compounds are selected
from the group consisting of a polynucleotide and a pharmaceutical
composition. Further provided are methods for detecting the micelle
composition.
Inventors: |
Mohapatra; Subhra; (Tampa,
FL) ; Mohapatra; Shyam S.; (Tampa, FL) ;
Howell; Mark Christian; (Tampa, FL) ; Dixit; Suraj
Kishore; (Tampa, FL) ; Wang; Chunyan; (Tampa,
FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mohapatra; Subhra
Mohapatra; Shyam S.
Howell; Mark Christian
Dixit; Suraj Kishore
Wang; Chunyan |
Tampa
Tampa
Tampa
Tampa
Tampa |
FL
FL
FL
FL
FL |
US
US
US
US
US |
|
|
Assignee: |
University of South Florida (A
Florida Non-Profit Corporation)
Tampa
FL
|
Family ID: |
49157871 |
Appl. No.: |
13/773749 |
Filed: |
February 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61602384 |
Feb 23, 2012 |
|
|
|
Current U.S.
Class: |
424/493 ;
514/44R |
Current CPC
Class: |
A61K 47/6909 20170801;
A61K 49/1821 20130101; A61K 49/1809 20130101; A61K 9/1075 20130101;
A61K 31/7088 20130101; A61K 9/167 20130101 |
Class at
Publication: |
424/493 ;
514/44.R |
International
Class: |
A61K 9/16 20060101
A61K009/16; A61K 31/7088 20060101 A61K031/7088 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] This invention was made with government support under grants
1R41CA139785 and 5R01CA152005-01 from the National Institutes of
Health. The U.S. government has certain rights in this invention.
Claims
1. A micelle composition comprising a micelle and either or both of
a pharmaceutical core and a polynucleotide, wherein the micelle
comprises a polyethylene glycol-phosphatidyl ethanolamine (PEG-PE),
a DC-cholesterol, and a dioleoylphosphatidyl-ethanolamine
(DOPE).
2. The micelle composition of claim 1, comprising a micelle and a
polynucleotide, wherein the micelle comprises a PEG-PE, a
DC-cholesterol, and a DOPE, and wherein the polynucleotide is
coated onto the micelle.
3. The micelle composition of claim 2, further comprising a
hydrophobic manganese-oleate core.
4. The micelle composition of claim 1, comprising a micelle and a
pharmaceutical core, wherein the micelle comprises a PEG-PE, a
DC-cholesterol, and a DOPE.
5. The micelle composition of claim 1, wherein the PEG has an
average molecular weight of between approximately 1800 Da and 2300
Da.
6. The micelle composition of claim 5, wherein the PEG-PE, the
DC-cholesterol, and the DOPE, comprise approximately 2%, 66%, and
32%, respectively, of the micelle.
7. The micelle composition of claim 1, wherein the micelle consists
essentially of a PEG-PE, a DC-cholesterol, and a DOPE.
8. The micelle composition of claim 7, wherein the PEG-PE, the
DC-cholesterol, and the DOPE, comprise approximately 2%, 66%, and
32%, respectively, of the micelle.
9. The micelle composition of claim 8, wherein the PEG has an
average molecular weight of between approximately 1800 Da and 2300
Da.
10. A method of administering one or more compounds to a cell
comprising, administering to the cell a micelle composition
comprising 1) a polyethylene glycol-phosphatidyl ethanolamine
(PEG-PE), a DC-cholesterol, and a dioleoylphosphatidyl-ethanolamine
(DOPE), and 2) the one or more compounds, wherein the compounds are
selected from the group consisting of a polynucleotide and a
pharmaceutical composition.
11. The method of claim 10, wherein the PEG has an average
molecular weight of between approximately 1800 Da and 2300 Da.
12. The method of claim 10, wherein the PEG-PE, the DC-cholesterol,
and the DOPE, comprise approximately 2%, 66%, and 32%,
respectively, of the micelle.
13. The method of claim 10, wherein the one or more compounds is a
polynucleotide, and the polynucleotide is coated onto the
micelle.
14. The method of claim 13, further comprising detecting the
micelle composition, wherein the micelle composition further
comprises a hydrophobic manganese-oleate core, and wherein the
micelle composition is detected using magnetic resonance imaging
technology.
15. The method of claim 10, wherein the one or more compounds is a
pharmaceutical and the micelle composition comprises a
pharmaceutical core.
16. The method of claim 10, wherein the one or more compounds are a
polynucleotide and a pharmaceutical, and wherein the polynucleotide
is coated onto the micelle and the micelle composition comprises a
pharmaceutical core.
17. The method of claim 10, wherein the cell is a lung cell in a
subject and the micelle composition is administered to the subject
intranasally.
18. The method of claim 10, wherein the micelle consists
essentially of a PEG-PE, a DC-cholesterol, and a DOPE.
19. The method of claim 18, wherein the PEG-PE, the DC-cholesterol,
and the DOPE, comprise approximately 2%, 66%, and 32%,
respectively, of the micelle.
20. The method of claim 19, wherein the PEG has an average
molecular weight of between approximately 1800 Da and 2300 Da.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional Patent Application Ser. No. 61/602,384 filed on Feb.
23, 2012.
BACKGROUND
[0003] Advances in nanoparticle technology have allowed the
development of multifunctional nanoparticles for cancer detection,
therapy, and treatment monitoring. Their numerous advantages
include cell-targeted delivery to minimize the amount of drug
needed to achieve a therapeutic dose [M. Schmitt-Sody et al.,
Clinical Cancer Research: An Official Journal of the American
Association for Cancer Research 2003 9, 2335], increased
bioavailability especially for hydrophobic drugs, reduced drug
toxicity [X. H. Peng et al., ACS Nano 2011, 5, 9480], enhanced
mucosal delivery that decreases first-pass metabolism [J. C. Sung,
B. L. Pulliam, & D. A. Edwards, Trends in Biotechnology 2007,
25, 563], controllable timing of drug delivery (slow-sustained,
pulsatile or stimulus-responsive) [I. Kim et al., Biomaterials
2012, 33, 5574; X. Shuai et al., Journal of Controlled Release:
Official Journal of the Controlled Release Society 2004, 98, 415;
H. Meng et al., ACS Nano 2011, 5, 4131], and the capacity to
combine drugs and imaging agents in the same particle [M. M.
Yallapu et al., Pharmaceutical Research 2010, 27, 2283; R. Kumar et
al., Theranostics 2012, 714; J. Shin et al., Angew Chem Int Ed Engl
2009, 48, 321]. Scalability, safety, and cost remain the most
formidable challenges in taking multifunctional nanoparticles from
the bench to clinical trials.
[0004] Magnetic resonance imaging (MRI) is one of the most widely
used noninvasive imaging and diagnostic techniques. It provides
detailed anatomical images of the body and is excellent for imaging
soft tissues. Contrast agents work by altering the T1, T2, or T1/T2
relaxation times of nearby protons. Positive contrast agents appear
brighter on the MRI owing to an increase in T1 signal intensity
caused by a reduction in the T1 relaxation times [E. C. Cho et al.,
Trends in Molecular Medicine 2010, 16, 561]. Superparamagnetic iron
oxide nanoparticles have been extensively studied for use in T2
contrast imaging in conjunction with a diverse array of
nanotherapeutics [Y. Ling et al., Biomaterials 2011, 32, 7139; S.
Laurent et al., Chemical Reviews 2008, 108, 2064; R. Rastogi et
al., Colloids and Surfaces B, Biointerfaces 2011, 82, 160].
Previously, we reported a unique formulation of
chitosan-polyethyleneimine nanoparticles with iron oxide in the
core for imaging together with a plasmid for gene delivery [C. Wang
et al., Journal of Controlled Release: Official Journal of the
Controlled Release Society, 2012]. However, since iron oxide is a
relatively poor T2-type MRI contrast agent for the lung [H. B. Na
et al., Angew Chem Int Ed Engl 2007, 46, 5397], there is a need to
develop nanoparticles containing T1 contrast agents for better lung
imaging that can also be used for drug delivery in lung
diseases.
[0005] Currently, T1 MRI utilizes predominantly gadolinium-
(Gd-)based contrast agents because of the large magnetic moment of
Gd.sup.3+ due to its seven unpaired electrons and slow electronic
relaxation time [D. Pan et. al., Wiley Interdisciplinary Reviews:
Nanomedicine and Nanobiotechnology, 2011, 3(2), 162; D. Pan et al.,
Tetrahedron 2011, 67, 8431]. The high toxicity of Gd.sup.3+,
however, requires that these contrast agents always be given in a
chelated form. Despite this, several cases of nephrogenic systemic
fibrosis (NSF) have been reported in patients receiving
Gd-containing contrast agents [M. R. Prince et al., Radiographics:
A Review Publication of the Radiological Society of North America,
Inc., 2009, 29, 1565; M. A. Sieber et al., Journal of Magnetic
Resonance Imaging: JMRI 2009, 30, 1268]. Hence, alternatives to
Gd-containing T1 contrast agents are needed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 shows a schematic representation of multi-functional
lipid-micellar nanoparticles. PL-1=payload 1; PL-2=payload 2.
[0007] FIG. 2 (A-C) shows cell uptake, viability, and in vivo
biodistribution of M-LMNs. (A) HEK293 cells were incubated with
M-LMNs (10 .mu.g/ml) for 4 hours and cell uptake was determined by
laser scanning confocal microscopy; z-stacked images of HEK293
cells showing uptake of rhodamine-conjugated M-LMNs. 1000.times.
magnification is shown. (B) Effects of M-LMN exposure in HEK293
cells. HEK293 cells were incubated for 72 hours with various
concentrations of M-LMNs and cell viability was assessed. (C) In
vivo biodistribution of Cy5.5-M-LMNs. Groups of mice (n=4) were
injected intravenously (IV) or intranasally (IN) with Cy5.5 M-LMNs
and at 24, 48 and 96 hours after administration, the Cy 5.5 levels
were quantitated by Xenogen imaging. Control mice received PBS
alone (IN). Relative fluorescent intensity per mg tissue is
shown.
[0008] FIG. 3 (A-B) shows (A) Gel electrophoresis (1% Agarose) of
the complexes of M-LMNs and DNA at different LMN:DNA weight ratios.
(B) Gel electrophoresis (0.8% Agarose) of complexes of M-LMNs and
DNA after exposure to DNase I.
[0009] FIG. 4 (A-D) shows the gene delivery potential of M-LMNs.
HEK293 cells were transfected with M-LMNs complexed with ptd DNA
encoding red-fluorescent protein (RFP). Transfection efficiency was
monitored by fluorescent microscopy. RFP images (upper panel) and
merge of fluorescent images (20.times. magnification) and
phase-contrast (bottom panel) are shown. (A-B) Various ratios of
M-LMN:DNA (wt/wt) were incubated with HEK293 cells for 48 hours.
The transfected cells were counted with imageJ and the percent
transfection in groups was compared with Graphpad Prism. 5:1 vs
10:1 *p<0.05. (C-D) Nanocomplexes of M-LMN:DNA wt/wt; 5:1 were
incubated for the indicated length of time. The transfected cells
were marked and counted using ImageJ software. The groups were
statistically compared in Graphpad Prism. 24 hours versus 48 hours
**p<0.01; 48 hours versus 96 hours ***p<0.001.
[0010] FIG. 5 (A-D) shows the MRI potential of M-LMNs containing
different concentrations of Mn.sup.2+. Two hundred .mu.l aliquots
of indicated concentrations of M-LMNs were added to a 96-well plate
in duplicate. T1 relaxometry map derived from the multi-TE T1
measurements, (A) Visual and (B) quantitative T1 MRI contrast is
shown. 50 .mu.l of a 0.7 mM Mn solution of M-LMNs were injected
intranasally to mice. After one hour the lungs were collected and
imaged ex vivo using MRI. (C) Visual and (D) quantitative T1 MRI
contrast are shown.
[0011] FIG. 6 (A-F) shows the cellular uptake, viability and in
vivo biodistribution of D-LMNs. (A) TEM of D-LMNs; scale bar=100
nm. (B) Laser scanning confocal microscopic images (1000.times.
magnification) (z-stacked) of uptake of D-LMNs by HEK293 cell. (C)
Release of DOX from D-MLNs in PBS at pH 7.3 and pH 5.4 as a
percentage of total encapsulated DOX. Free DOX was used as control.
(D) Effect of D-LMNs on viability of LLC1 cells. Cells were
incubated for 72 hours with various concentrations of M-LMNs or
D-LMNs, and viability was assessed by Presto Blue assay. (E)
Comparison of exposure of D-LMNs with free DOX in LLC1 cells. (F)
In vivo bio-distribution of D-LMNs. Groups of mice (n=4) were
treated intranasally with six rounds of D-LMNs over a two-week
period, the DOX levels in each organ were quantitated by Xenogen
imaging. Control mice received PBS. Relative fluorescent intensity
per mg tissue is shown.
[0012] FIG. 7 (A-F) shows cellular uptake, viability, gene
transduction, and imaging potential of DM-LMNs. (A) Laser scanning
confocal microscopic images (630.times. magnification; z-stacked)
of HEK293 cells showing uptake of D-LMNs. (B) Treatment of LLC1
cells with D-LMNs compared to free DOX. LLC1 cells were incubated
for 72 hours with various concentrations of D-LMNs or free DOX and
cell viability was determined. (C-D) Transfection potential of
DM-LMNs. HEK293 cells were transfected with DM-LMNs complexed with
ptdTomato plasmid DNA at wt/wt ratios of 5:1 or 10:1. Transfection
efficiency was determined by fluorescence microscopy. Red
fluorescent protein (upper panel) and the merge of RFP and the
phase-contrast image (bottom panel) (200.times. magnification) are
shown. Transfected cells were counted separately using ImageJ
software. The percent of transfected cells were compared with
Graphpad Prism. *p<0.05. (E) Simultaneous green fluorescent
protein transfection and DOX delivery by DM-LMNs in HEK293 cells.
(F) In vivo EGFP-DNA transfection by M-LMNs (a-b) and simultaneous
EGFP-DNA transfection and DOX delivery by DM-LMNs (c-e) in mouse
lungs (1000.times. magnification) after 72 hours.
DETAILED DESCRIPTION
[0013] Provided herein is a micelle composition comprising a
polyethylene glycol (PEG), a DC-cholesterol, and a
dioleoylphosphatidyl-ethanolamine (DOPE) and either or both a
pharmaceutical compound core and a polynucleotide coating. Also
provided herein is a method of administering one or more compounds
to a cell comprising, contacting the cell with a micelle
composition comprising 1) a polyethylene glycol-phosphatidyl
ethanolamine (PEG-PE), a DC-cholesterol, and a
dioleoylphosphatidyl-ethanolamine (DOPE), and 2) the one or more
compounds, wherein the compounds are selected from the group
consisting of a polynucleotide and a pharmaceutical composition.
Further provided are methods for detecting the micelle composition.
Term definitions used in the specification and claims are as
follows.
[0014] Definitions
[0015] As used in the specification and claims, the singular form
"a," "an," and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes
a plurality of cells, including mixtures thereof.
[0016] The term "active derivative" and the like means a modified
PEG-PE, DC-cholesterol, or DOPE composition that retains an ability
to form a micelle that protects a polynucleotide from nuclease
digestion. Assays for testing the ability of an active derivative
to perform in this fashion are known to those of ordinary skill in
the art.
[0017] When referring to a subject or patient, the term
"administering" refers to an administration that is oral, topical,
intravenous, subcutaneous, transcutaneous, transdermal,
intramuscular, intra joint, parenteral, intra-arteriole,
intradermal, intraventricular, intracranial, intraperitoneal,
intralesional, intranasal, rectal, vaginal, by inhalation or via an
implanted reservoir. The term "parenteral" includes subcutaneous,
intravenous, intramuscular, intra-articular, intra-peritoneal,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional, and intracranial injections or infusion techniques.
Is some embodiments, the administration is intranasal.
[0018] The term "antibody" is used in the broadest sense and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, and multispecific
antibodies (e.g., bispecific antibodies). Antibodies (Abs) and
immunoglobulins (Igs) are glycoproteins having the same structural
characteristics. While antibodies exhibit binding specificity to a
specific target, immunoglobulins include both antibodies and other
antibody-like molecules that lack target specificity. Native
antibodies and immunoglobulins are usually heterotetrameric
glycoproteins of about 150,000 Daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each heavy
chain has at one end a variable domain (V.sub.H) followed by a
number of constant domains. Each light chain has a variable domain
at one end (V.sub.L) and a constant domain at its other end. An
antibody "specific for" another substance binds, is bound by, or
forms a complex with that substance.
[0019] The term "antibody fragment" refers to a portion of a
full-length antibody, generally the target binding or variable
region. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2 and Fv fragments. The phrase "functional fragment or
analog" of an antibody is a compound having qualitative biological
activity in common with a full-length antibody. For example, a
functional fragment or analog of an anti-IgE antibody is one which
can bind to an IgE immunoglobulin in such a manner so as to prevent
or substantially reduce the ability of such a molecule from having
the ability to bind to the high affinity receptor, Fc.epsilon.RI.
As used herein, "functional fragment" with respect to antibodies
refers to Fv, F(ab) and F(ab').sub.2 fragments. The Fab fragment
contains the constant domain of the light chain and the first
constant domain (CH1) of the heavy chain. Fab' fragments differ
from Fab fragments by the addition of a few residues at the
carboxyl terminus of the heavy chain CH1 domain including one or
more cysteines from the antibody hinge region. F(ab') fragments are
produced by cleavage of the disulfide bond at the hinge cysteines
of the F(ab').sub.2 pepsin digestion product. Additional chemical
couplings of antibody fragments are known to those of ordinary
skill in the art. An "Fv" fragment is the minimum antibody fragment
which contains a complete target recognition and binding site. This
region consists of a dimer of one heavy and one light chain
variable domain in a tight, non-covalent association
(V.sub.H-V.sub.L dimer). It is in this configuration that the three
CDRs of each variable domain interact to define a target binding
site on the surface of the V.sub.H-V.sub.L dimer. Collectively, the
six CDRs confer target binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three CDRs specific for a target) has the ability to recognize
and bind target, although at a lower affinity than the entire
binding site. "Single-chain Fv" or "sFv" antibody fragments
comprise the V.sub.H and V.sub.L domains of an antibody, wherein
these domains are present in a single polypeptide chain. Generally,
the Fv polypeptide further comprises a polypeptide linker between
the V.sub.H and V.sub.L domains, which enables the sFv to form the
desired structure for target binding.
[0020] As used herein, the terms "cancer," "cancer cells,"
"neoplastic cells," "neoplasia," "tumor," and "tumor cells" (used
interchangeably) refer to cells which exhibit relatively autonomous
growth, so that they exhibit an aberrant growth phenotype
characterized by a significant loss of control of cell
proliferation (i.e., de-regulated cell division). Neoplastic cells
can be malignant or benign. A metastatic cell or tissue means that
the cell can invade and destroy neighboring body structures. The
cancer can be selected from astrocytoma, adrenocortical carcinoma,
appendix cancer, basal cell carcinoma, bile duct cancer, bladder
cancer, bone cancer, brain cancer, brain stem glioma, breast
cancer, cervical cancer, colon cancer, colorectal cancer, cutaneous
T-cell lymphoma, ductal cancer, endometrial cancer, ependymoma,
Ewing sarcoma, esophageal cancer, eye cancer, gallbladder cancer,
gastric cancer, gastrointestinal cancer, germ cell tumor, glioma,
hepatocellular cancer, histiocytosis, Hodgkin lymphoma,
hypopharyngeal cancer, intraocular melanoma, Kaposi sarcoma, kidney
cancer, laryngeal cancer, leukemia, liver cancer, lung cancer,
lymphoma, macroglobulinemia, melanoma, mesothelioma, mouth cancer,
multiple myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin
lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer,
parathyroid cancer, penile cancer, pharyngeal cancer, pituitary
cancer, prostate cancer, rectal cancer, renal cell cancer,
retinoblastoma, rhabdomyosarcoma, sarcoma, skin cancer, small cell
lung cancer, small intestine cancer, squamous cell carcinoma,
stomach cancer, T-cell lymphoma, testicular cancer, throat cancer,
thymoma, thyroid cancer, trophoblastic tumor, urethral cancer,
uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer and
Wilms tumor. In some embodiments, the cancer is prostate
cancer.
[0021] It should be understood that the term "coating" describes
the method of applying a compound such as a polynucleotide to a
pre-formed micelle and does not necessarily indicate the ultimate
location of the compound on the exterior of the micelle. It should
be further understood that the term "coating" does not require a
complete coverage of the coated object and that partial coverage is
encompassed by the term.
[0022] As used herein, the term "comprising" is intended to mean
that the compositions and methods include the recited elements, but
not excluding others. "Consisting essentially of," when used to
define compositions and methods, shall mean excluding other
elements of any essential significance to the combination. Thus, a
composition consisting essentially of the elements as defined
herein would not exclude trace contaminants from the isolation and
purification method and pharmaceutically acceptable carriers, such
as phosphate buffered saline, preservatives, and the like.
"Consisting of" shall mean excluding more than trace elements of
other ingredients and substantial method steps for administering
the compositions of this invention. Embodiments defined by each of
these transition terms are within the scope of this invention.
[0023] An "effective amount" is an amount sufficient to effect
beneficial or desired results. An effective amount can be
administered in one or more administrations, applications or
dosages.
[0024] As used herein, "expression" refers to the process by which
polynucleotides are transcribed into mRNA and/or the process by
which the transcribed mRNA is subsequently translated into
peptides, polypeptides, or proteins. If the polynucleotide is
derived from genomic DNA, expression may include splicing of the
mRNA in a eukaryotic cell. "Overexpression" as applied to a gene
refers to the overproduction of the mRNA transcribed from the gene
or the protein product encoded by the gene, at a level that is 2.5
times higher, preferably 5 times higher, more preferably 10 times
higher, than the expression level detected in a control sample.
[0025] A "gene" refers to a polynucleotide containing at least one
open reading frame that is capable of encoding a particular
polypeptide or protein after being transcribed and translated. Any
of the polynucleotide sequences described herein may be used to
identify larger fragments or full-length coding sequences of the
gene with which they are associated. Methods of isolating larger
fragment sequences are known to those of skill in the art, some of
which are described herein.
[0026] A "gene product" refers to the amino acid (e.g., peptide or
polypeptide) generated when a gene is transcribed and
translated.
[0027] "Humanized" forms of non-human (e.g. murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
target-binding subsequences of antibodies) that contain minimal
sequence derived from non-human immunoglobulin. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody may also comprise at least a portion of an immunoglobulin
constant region (Fc), typically that of a human immunoglobulin
template chosen.
[0028] The term "isolated" means separated from constituents,
cellular and otherwise, in which the polynucleotide, peptide,
polypeptide, protein, antibody, or fragments thereof are normally
associated with in nature. In one aspect of this invention, an
isolated polynucleotide is separated from the 3' and 5' contiguous
nucleotides with which it is normally associated with in its native
or natural environment, e.g., on the chromosome. As is apparent to
those of skill in the art, a non-naturally occurring
polynucleotide, peptide, polypeptide, protein, antibody, or
fragments thereof does not require "isolation" to distinguish it
from its naturally occurring counterpart. In addition, a
"concentrated," "separated," or "diluted" polynucleotide, peptide,
polypeptide, protein, antibody, or fragments thereof is
distinguishable from its naturally occurring counterpart in that
the concentration or number of molecules per volume is greater than
"concentrated" or less than "separated" than that of its naturally
occurring counterpart. A polynucleotide, peptide, polypeptide,
protein, antibody, or fragments thereof, which differs from the
naturally occurring counterpart in its primary sequence or for
example, by its glycosylation pattern, need not be present in its
isolated form since it is distinguishable from its naturally
occurring counterpart by its primary sequence, or alternatively, by
another characteristic such as glycosylation pattern. Although not
explicitly stated for each of the inventions disclosed herein, it
is to be understood that all of the above embodiments for each of
the compositions disclosed below and under the appropriate
conditions are provided by this invention. Thus, a non-naturally
occurring polynucleotide is provided as a separate embodiment from
the isolated naturally occurring polynucleotide. A protein produced
in a bacterial cell is provided as a separate embodiment from the
naturally occurring protein isolated from a eukaryotic cell in
which it is produced in nature.
[0029] As used herein, the term "micelle" refers to a single layer
aggregation of molecules wherein hydrophobic portions of the
molecules comprise the interior of the aggregation and hydrophilic
portions of the molecules comprise the exterior of the aggregation.
Accordingly, the term "micelle" refers herein to the molecules that
aggregate to form the micelle, for example, polyethylene
glycol-phosphatidyl ethanolamine (PEG-PE),
3.beta.-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol
(DC-cholesterol), and dioleoylphosphatidyl-ethanolamine (DOPE). The
term "single layer" excludes bilayer compositions such as liposomes
from the definition of a micelle. Liposomes are structurally
different from micelles in that they have a bilayer membrane. This
bilayer nature provides benefits for drug delivery that are not
found in single layer aggregations such as micelles. Further, when
a compound resides in a "micelle core," that compound resides in
the interior of the micelle aggregation. When a compound is coated
onto the exterior of a micelle, that compound can ultimately reside
in the exterior of the micelle aggregation, reside in the interior
of the micelle aggregation, or reside in both the exterior and
interior portions of the micelle aggregation. As described herein,
"micelle compositions" contain materials in addition to the micelle
itself, such as core compounds and coated compounds.
[0030] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
target site. Furthermore, in contrast to conventional (polyclonal)
antibody preparations which typically include different antibodies
directed against different determinants (epitopes), each monoclonal
antibody is directed against a single determinant on the target. In
addition to their specificity, monoclonal antibodies are
advantageous in that they may be synthesized by the hybridoma
culture, uncontaminated by other immunoglobulins. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies
for use with the present invention may be isolated from phage
antibody libraries using well-known techniques. The parent
monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method or may be made by
recombinant methods.
[0031] A "pharmaceutical composition" is intended to include the
combination of an active agent with a carrier, inert or active,
making the composition suitable for diagnostic or therapeutic use
in vitro, in vivo or ex vivo.
[0032] The term "pharmaceutically acceptable carrier or excipient"
means a carrier or excipient that is useful in preparing a
pharmaceutical composition that is generally safe, non-toxic and
neither biologically nor otherwise undesirable and includes a
carrier or excipient that is acceptable for veterinary use as well
as human pharmaceutical use. A "pharmaceutically acceptable carrier
or excipient" as used in the specification and claims includes both
one and more than one such carrier or excipient. As used herein,
the term "pharmaceutically acceptable carrier" encompasses any of
the standard pharmaceutical carriers, such as a phosphate buffered
saline solution, water, emulsions, such as an oil/water or
water/oil emulsion, and various types of wetting agents. The
compositions also can include stabilizers and preservatives.
[0033] The term "pharmaceutically acceptable salts" refers to any
acid or base addition salt whose counter-ions are non-toxic to the
subject to which they are administered in pharmaceutical doses of
the salts. Specific examples of pharmaceutically acceptable salts
are provided below.
[0034] The terms "pharmaceutically effective amount,"
"therapeutically effective amount," or "therapeutically effective
dose" refer to the amount of a compound that will elicit the
biological or medical response of a tissue, system, animal, or
human that is being sought by the researcher, veterinarian, medical
doctor or other clinician.
[0035] The term "therapeutically effective amount" includes that
amount of a compound that, when administered, is sufficient to
prevent development of, or alleviate to some extent, one or more of
the symptoms of the condition or disorder being treated. The
therapeutically effective amount will vary depending on the
compound, the disorder or conditions and their severity, the route
of administration, time of administration, rate of excretion, drug
combination, judgment of the treating physician, dosage form, and
the age, weight, general health, sex and/or diet of the subject to
be treated.
[0036] The terms "polynucleotide" and "oligonucleotide" are used
interchangeably and refer to a polymeric form of nucleotides of any
length, either deoxyribonucleotides or ribonucleotides, or analogs
thereof. Polynucleotides may have any three-dimensional structure
and may perform any function, known or unknown. The following are
non-limiting examples of polynucleotides: a gene or gene fragment,
exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA,
siRNA, ribozymes, cDNA, recombinant polynucleotides, branched
polynucleotides, plasmids, vectors, isolated DNA of any sequence,
isolated RNA of any sequence, polynucleotide probes, and primers. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and nucleotide analogs. If present,
modifications to the nucleotide structure may be imparted before or
after assembly of the polymer. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after polymerization, such as by conjugation with
a labeling component. The term also refers to both double- and
single-stranded molecules. Unless otherwise specified or required,
any embodiment of this invention that is a polynucleotide
encompasses both the double-stranded form and each of two
complementary single-stranded forms known or predicted to make up
the double-stranded form.
[0037] A polynucleotide is composed of a specific sequence of four
nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine
(T); and uracil (U) for thymine (T) when the polynucleotide is RNA.
Thus, the term "polynucleotide sequence" is the alphabetical
representation of a polynucleotide molecule. This alphabetical
representation can be input into databases in a computer having a
central processing unit and used for bioinformatics applications
such as functional genomics and homology searching.
[0038] The term "polypeptide" is used in its broadest sense to
refer to a compound of two or more subunit amino acids, amino acid
analogs, or peptidomimetics. The subunits may be linked by peptide
bonds. In another embodiment, the subunit may be linked by other
bonds, e.g. ester, ether, etc. As used herein the term "amino acid"
refers to either natural and/or unnatural or synthetic amino acids,
including glycine and both the D or L optical isomers, and amino
acid analogs and peptidomimetics. A peptide of three or more amino
acids is commonly called an oligopeptide if the peptide chain is
short. If the peptide chain is long, the peptide is commonly called
a polypeptide or a protein.
[0039] "Selectively binds" refers to a non-specific binding event
as determined by an appropriate comparative control. Binding is
selective when the binding is at least 10, 30, or 40 times greater
than that of background binding in the comparative control.
[0040] A "subject," "individual" or "patient" is used
interchangeably herein, which refers to a vertebrate, preferably a
mammal, more preferably a human. Mammals include, but are not
limited to, murines, simians, humans, farm animals, sport animals,
and pets.
[0041] "Transformation" of a cellular organism with DNA means
introducing DNA into an organism so that the DNA is replicable,
either as an extrachromosomal element or by chromosomal
integration. "Transfection" of a cellular organism with DNA refers
to the taking up of DNA, e.g., an expression vector, by the cell or
organism whether or not any coding sequences are in fact expressed.
The terms "transfected host cell" and "transformed host cell" refer
to a cell in which DNA was introduced. The cell is termed "host
cell" and it may be either prokaryotic or eukaryotic. Typical
prokaryotic host cells include various strains of E. coli. Typical
eukaryotic host cells are mammalian, such as Chinese hamster ovary
cells or cells of human origin. The introduced DNA sequence may be
from the same species as the host cell of a different species from
the host cell, or it may be a hybrid DNA sequence, containing some
foreign and some homologous DNA.
[0042] The term "vector" means a DNA construct containing a DNA
sequence which is operably linked to a suitable control sequence
capable of effecting the expression of the DNA in a suitable host.
Such control sequences include a promoter to effect transcription,
an optional operator sequence to control such transcription, a
sequence encoding suitable mRNA ribosome binding sites, and
sequences which control the termination of transcription and
translation. The vector may be a plasmid, a phage particle, or
simply a potential genomic insert. Once transformed into a suitable
host, the vector may replicate and function independently of the
host genome or may, in some instances, integrate into the genome
itself. In the present specification, "plasmid" and "vector" are
sometimes used interchangeably, as the plasmid is the most commonly
used form of vector. However, the invention is intended to include
such other forms of vectors which serve equivalent function as and
which are, or become, known in the art.
[0043] Accordingly, provided herein is a micelle composition
comprising a polyethylene glycol-phosphatidyl ethanolamine
(PEG-PE), a 3.beta.-[N-(N',N'-dimethylaminoethane)-carbamoyl]
cholesterol (DC-cholesterol), and a
dioleoylphosphatidyl-ethanolamine (DOPE) and either or both a
pharmaceutical compound core and a polynucleotide coating. In some
embodiments, the micelle composition further comprises an imaging
contrast agent. These cationic lipid micellar nanoparticles are
referred to herein as "LMNs." FIG. 1 provides a general schematic
of the micelle composition.
[0044] The polyethylene glycol-phosphatidyl ethanolamine (PEG-PE)
found in the micelle composition can be of any molecular weight
that allows for formation of a micelle with DC-cholesterol and
DOPE, The PEG-PE compound includes PEG molecules having an average
molecular weight between approximately 570-630 Da (PEG 600),
720-880 Da (PEG 800), 950-1050 Da (PEG 1000), 1800-2200 Da (PEG
2000), 2700-3300 Da (PEG 3000), 3500-4500 Da (PEG 4000), or
5000-7000 Da (PEG 6000). In one embodiment, the PEG-PE compound
comprises PEG molecules having an average molecular weight between
approximately 1800-2200 Da. Accordingly, included in the present
invention is a micelle composition comprising
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000]. It should be understood that most PEG compounds
include molecules with a distribution of molecular weights (i.e.,
they are polydisperse). The size distribution can be characterized
statistically by its weight average molecular weight (Mw) and its
number average molecular weight (Mn), the ratio of which is called
the polydispersity index (Mw/Mn). MW and Mn can be measured by mass
spectrometry.
[0045] It should be understood that the PEG-PE, DC-cholesterol, and
DOPE can be present in any ratio or percentage that allows for
micelle formation. In some embodiments, the micelle comprises
between approximately 1-10%, 50-75%, and 15-40% of the PEG-PE, the
DC-cholesterol, and the DOPE, respectively. Accordingly, the
micelle composition can comprise approximately 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%. 9%, or 10% of a PEG-PE. The micelle composition can
comprise approximately 50%, 55%, 60%, 65%, 70%, or 75% of a
DC-cholesterol. The micelle composition can comprise approximately
15%, 20%, 25%, 30%, 35%, or 40% of a DOPE. In one embodiment, the
micelle comprises between approximately 2%, 66%, and 32% of a
PEG-PE, a DC-cholesterol, and a DOPE, respectively.
[0046] The micelle compositions provided herein comprise either or
both a pharmaceutical compound core and a polynucleotide coating.
It should be understood that the pharmaceutical can be any compound
that is hydrophobic or that can be made to be hydrophobic. In one
embodiment, the pharmaceutical is a cancer chemotherapy compound,
and in certain further embodiments, the pharmaceutical is
doxorubicin. A micelle comprises a pharmaceutical compound core
when the pharmaceutical resides in the core, or interior, of the
micelle.
[0047] Micelle compositions that comprise a "polynucleotide
coating" refer to pre-formed micelle compositions to which
polynucleotides are applied. When a polynucleotide is coated onto
the exterior of a micelle, that polynucleotide can ultimately
reside in the exterior of the micelle aggregation, reside in the
interior of the micelle aggregation, or reside in both the exterior
and interior portions of the micelle aggregation. The
polynucleotides can be of any length, either deoxyribonucleotides
or ribonucleotides, or analogs thereof. Polynucleotides may have
any three-dimensional structure and may perform any function, known
or unknown. The following are non-limiting examples of
polynucleotides: a gene or gene fragment, exons, introns, messenger
RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, ribozymes, cDNA,
recombinant polynucleotides, branched polynucleotides, plasmids,
vectors, isolated DNA of any sequence, isolated RNA of any
sequence, polynucleotide probes, and primers. The micelle
compositions provided herein can comprise any amount of
polynucleotide. In some embodiments, the polynucleotide coating is
applied to a micelle at a micelle:polynucleotide molecular weight
ratio of approximately 15:1, 10:1, 5:1, 2:1, or 1:1.
[0048] In some embodiments, the micelle composition further
comprises a hydrophobic contrast imaging agent located in its core.
Contrast imaging agents include, but are not limited to, T1
magnetic resonance imaging (MRI) agents and T2 MRI agents. In one
embodiment, the contrast imaging agent is a T1 MRI agent. T1 MRI
agents include manganese (Mn), and the present invention includes
micelles having a hydrophobic manganese core. In some embodiments,
the micelle composition comprises a manganese-oleate core.
[0049] The nonlanthanide metal manganese (Mn) is paramagnetic, has
five unpaired electrons in its bivalent state, and is a natural
cellular constituent as a cofactor for enzymes and receptors [D.
Pan et al., Wiley Interdisciplinary Reviews: Nanomedicine and
Nanobiotechnology, 2011, 3(2), 162; D. Pan et al., Tetrahedron
2011, 67, 8431]. The intrinsic properties of Mn include high spin
number, long electronic relaxation time, and labile water exchange.
Though Mn-containing contrast agents are FDA approved for clinical
use [D. Pan et al., Wiley Interdisciplinary Reviews: Nanomedicine
and Nanobiotechnology, 2011, 3(2), 162], Mn can be toxic at the
high levels required to offset the short plasma half-life of ionic
Mn [D. Pan et al., Tetrahedron 2011, 67, 8431; J. Y. Choi et al.,
Bioprocess and Biosystems Engineering 2010, 33 21]. In formulating
the micelles described herein, it was hypothesized that
sequestration of Mn within nanoparticles could possibly reduce the
risk of toxicity and overcome the problem of short plasma
half-life.
[0050] Accordingly, provided herein are micelle compositions
comprising a PEG-PE, a DC-cholesterol, and a DOPE and further
comprising a pharmaceutical compound core, a hydrophobic imaging
agent core, and/or a polynucleotide coating. In one embodiment, the
micelle composition comprises a PEG-PE, a DC-cholesterol, and a
DOPE and further comprises a pharmaceutical compound core. In
another embodiment, the micelle composition comprises a PEG-PE, a
DC-cholesterol, and a DOPE and further comprises a pharmaceutical
compound core and a polynucleotide coating. In yet another
embodiment, the micelle composition comprises a PEG-PE, a
DC-cholesterol, and a DOPE and further comprises a pharmaceutical
compound and hydrophobic imaging agent core and a polynucleotide
coating. In a still further embodiment, the micelle composition
comprises a PEG-PE, a DC-cholesterol, and a DOPE and further
comprises a polynucleotide coating and a hydrophobic imaging agent
core.
[0051] In some embodiments, the micelle composition further
comprises a ligand. A ligand is defined herein as any moiety that
facilitates binding of the compositions provided herein to a target
such as a cell. Ligands include, but are not limited to,
antibodies, adhesion molecules, lectins, integrins, and selectins.
When the ligand is an antibody, it can comprise approximately 1% of
the total composition weight (but is not limited to such amount).
In some embodiments, the ligand is an antibody specific for a
cancer cell.
[0052] The micelle compositions provided herein are useful for
administering polynucleotides, pharmaceutical compositions, and/or
MRI imaging agents to cells, and in particular, to cells in a
subject. The examples below describe in vitro MRI, cellular uptake,
transfection, cytotoxicity studies, and in vivo experiments in mice
which demonstrate that these cationic lipid nanoparticles act as a
T1 contrast agent and DNA/drug delivery vehicle. It was a
surprising finding of the present invention that the unique
combination of DOPE, DC-cholesterol and PEG-2000-PE yielded a high
gene transfection efficiency and drug uptake. When administered to
mice intranasally as nasal drops, the DM-LMN nanoparticles were
found mostly in the lungs, in marked contrast to other polymers,
making them an ideal candidate for lung cancer theranostics.
[0053] Accordingly, provided herein is a method of administering
one or more compounds to a cell comprising, administering to the
cell a micelle composition comprising 1) a PEG-PE, a
DC-cholesterol, and a DOPE, and 2) the one or more compounds,
wherein the compounds are selected from the group consisting of a
polynucleotide and a pharmaceutical composition. In some
embodiments, the administered micelle composition comprises a
polynucleotide coating as described above. When administering a
micelle composition comprising a polynucleotide, the method can
include transfecting and/or transforming a cell to which the
micelle composition is administered. In other embodiments, the
administered micelle composition comprises a pharmaceutical
compound core as described above. In still other embodiments, the
administered micelle composition comprises both a polynucleotide
coating and a pharmaceutical compound core.
[0054] The micelle composition can be administered to a cell in
vitro, in vivo, or ex vivo. In one embodiment, the micelle
composition is administered to a subject. When referring to a
subject or patient, the terms "administered" and "administering"
refer to an administration that is oral, topical, intravenous,
subcutaneous, transcutaneous, transdermal, intramuscular, intra
joint, parenteral, intra-arteriole, intradermal, intraventricular,
intracranial, intraperitoneal, intralesional, intranasal, rectal,
vaginal, by inhalation or via an implanted reservoir. The term
"parenteral" includes subcutaneous, intravenous, intramuscular,
intra-articular, intra-peritoneal, intra-synovial, intrasternal,
intrathecal, intrahepatic, intralesional, and intracranial
injections or infusion techniques. The methods can also comprise
placing a magnet proximal to a target cell or group of target cells
prior to, during, and/or after administration of the micelle
composition to a subject containing the cell(s). A target cell is
that cell to which delivery of the micelle composition is desired.
In some embodiments, the target cells are lung cells.
[0055] The examples below indicate that intranasal administration
of the micelle composition provided herein to a subject directs the
micelle composition to the lungs of the subject. Accordingly, in
some embodiments, the administration is intranasal. In still
further embodiments, the administration is intranasal and the cell
to which the micelle composition is delivered is a lung cell.
[0056] The examples below further indicate that the micelle
composition described herein can contain an MRI imaging agent,
which agent permits the visualization of the micelle composition
after it is administered to a subject. Accordingly, included herein
is a method of administering one or more compounds to a subject
comprising, administering to the subject a micelle composition
comprising 1) a PEG-PE, a DC-cholesterol, and a DOPE, 2) the one or
more compounds, wherein the compounds are selected from the group
consisting of a polynucleotide and a pharmaceutical composition,
and 3) an MRI imaging agent. In one embodiment, the MRI imaging
agent is a hydrophobic manganese-oleate core. Also provided herein
is a method of detecting the administration of one or more
compounds to a subject comprising, administering to the subject a
micelle composition comprising 1) a PEG-PE, a DC-cholesterol, and a
DOPE, and 2) the one or more compounds, wherein the compounds are
selected from the group consisting of a polynucleotide and a
pharmaceutical composition, and 3) an MRI imaging agent; and
detecting a location of the micelle composition in the subject
using magnetic resonance imaging technology. In one embodiment, the
administration of a polynucleotide is detected. In another
embodiment, the administration of a pharmaceutical compound is
detected.
[0057] It should be understood that the foregoing relates to
preferred embodiments of the present disclosure and that numerous
changes may be made therein without departing from the scope of the
disclosure. The disclosure is further illustrated by the following
examples, which are not to be construed in any way as imposing
limitations upon the scope thereof. On the contrary, it is to be
clearly understood that resort may be had to various other
embodiments, modifications, and equivalents thereof, which, after
reading the description herein, may suggest themselves to those
skilled in the art without departing from the spirit of the present
disclosure and/or the scope of the appended claims. All patents,
patent applications, and publications referenced herein are
incorporated by reference in their entirety for all purposes.
EXAMPLES
Example 1
Preparation and Characterization of Manganese LMNs (M-LMNs)
[0058] To prepare M-LMNs, Mn.sup.2+-oleate complexes were subjected
to thermal decomposition in a high boiling-point solvent that
produces MnO with a hydrophobic surface layer of oleic acid. To
create a hydrophilic exterior, phospholipid micelles encapsulating
these MnO nanoparticles were prepared by the thin-film hydration
method in which hydrophobic MnO nanoparticles were added to a
mixture of PEG-2000 PE, DC-cholesterol and DOPE dissolved in
chloroform. The particles were vacuum dried and the dry film was
swelled in water, sonicated and centrifuged to remove uncoated MnO
nanoparticles. The micelles coating the MnO nanoparticles are
composed of ingredients that have been FDA approved for use in
humans or have been used in clinical trials. The lipids DOPE and
DC-cholesterol have been used in clinical trials for the nasal
delivery of DNA to cystic fibrosis patients [D. R. Gill et al.,
Gene Therapy 1997, 4, 199; N. J. Caplen et al., Nature Medicine
1995, 1, 39; S. C. Hyde et al., Gene Therapy 2000, 7, 1156; P. G.
Middleton et al., The European Respiratory Journal: Official
Journal of the European Society for Clinical Respiratory Physiology
1994, 7, 442]. Lipid-conjugated PEG-2000 is an essential part of
the FDA-approved formulation Doxil.RTM. [Y. Barenholz, Journal of
Controlled Release: Official Journal of the Controlled Release
Society 2012, 160, 117].
[0059] More specifically, the following materials and methods were
used.
[0060] Materials: Manganese sulfate, sodium oleate, chloroform,
hexane, 1-octadecane, dichloromethane, triethylamine, and acetone
were all purchased from Sigma. PEG-2000 PE, DC-cholesterol, DOPE,
and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-lissamine
rhodamine B sulfonyl were all purchased from Avanti Polar Lipids.
Cy5.5 NHS was purchased from Invitrogen. All reagents were used
without further purification.
[0061] Synthesis of Mn-oleate complexes: Mn-oleate was prepared by
the method described previously with some modifications [J. Park et
al., Nature Materials 2004, 3, 891]. Manganese sulfate (2 g) and
sodium oleate (6.1 g) were dissolved in a combination of ethanol
(7.5 ml), hexane (17 ml), and distilled water (10 ml). The solution
was heated to 70.degree. C. with vigorous stirring overnight. The
solution was then transferred to a separatory funnel and the upper
organic layer containing the Mn-oleate complex was washed several
times using distilled water. The purified solution was allowed to
evaporate, producing a deep red waxy solid that was the
manganese-oleate complex.
[0062] Hydrophobic MnO nanoparticles were then prepared by the
method described previously with some modifications [J. Park et
al., Nature Materials 2004, 3, 891]. Manganese-oleate (1.3 g) was
dissolved in 1-octadecene (13.5 ml), and degassed at 70.degree. C.
for 1 hour under vacuum with vigorous stirring. The solution was
then purged with argon and heated to 300.degree. C. while stirring
under argon. As the temperature reached 300.degree. C., the
initially red solution turned transparent and then pale green. The
reaction was held at this temperature for 1 hour and 15 minutes,
then allowed to cool to room temperature after which
dichloromethane (20 ml) was added to improve the dispersibility of
the nanoparticles. Acetone (80 ml) was added to precipitate the
nanoparticles and the solution was centrifuged at 3,000 rpm
(835.times.g) at 4.degree. C. for 15 minutes. Supernatants were
discarded and the pellets were reconstituted in 20 ml of
dichloromethane. The above purification procedure was repeated two
more times to remove excess surfactant and solvent. The purified
MnO nanoparticles were dispersible in many organic solvents such as
dichloromethane and chloroform.
[0063] Preparation of MnO nanoparticles encapsulated in micelles:
MnO nanoparticles were encapsulated inside micelles using a
published procedure with some modifications [R. Kumar et al.,
Theranostics 2012, 714-722; Y. Namiki et al., Nature Nanotechnology
2009, 4, 598; B. Dubertret et al., Science 2002, 298, 1759].
PEG-2000 PE (0.1 mg, 2% of total), DC-cholesterol (7.9 .mu.M, 3.95
mg, 66% of total), and DOPE (2.6 .mu.M, 1.95 mg, 32% of total) were
added to 1.5 ml of chloroform. Then 3 mg (0.23 ml of stock
solution) of MnO nanoparticles were added to this solution. To
ensure complete solubilization, the reaction solution was sonicated
in a Branson 2510 sonicator for 20 minutes. The solution was then
left to evaporate overnight in a vacuum oven at 40.degree. C. The
dry film was heated at 80.degree. C. for 2 minutes. Then 2 ml of
water was added and the solution was again sonicated for 3 hours.
After the film was dissolved, the solution was centrifuged at
90,000 rpm (334,000.times.g) at 4.degree. C. for 2 hours to
separate filled micelles from empty ones. The pellet was
reconstituted in 1 ml of water and sonicated further for 30
minutes. The M-LMNs were filtered through a 0.45-micron syringe
filter and stored at 4.degree. C.
[0064] Chemical and physical characterization of nanoparticles:
FTIR spectra of oleic acid-coated MnO nanoparticles were obtained
using a Nicolet IR-100 spectrometer. A 20 .mu.l aliquot of the
oleic acid-coated MnO nanoparticles dispersed in chloroform was
dropped onto a disposable polyethylene IR card and the solvent was
evaporated under vacuum before taking the measurements. FTIR
spectrometry was performed on the MnO-oleate nanoparticles and
bands characteristic of oleic acid-coated hydrophobic MnO
nanoparticles were identified. The surface-bonded oleic acid was
confirmed by the presence of bands in the 2900 and 2850 cm.sup.-1
range, due to the C--H stretch, and a band at 1461 cm.sup.-1 (C--H
bending) [C. Wang et al., Journal of Controlled Release: Official
Journal of the Controlled Release Society, 2012].
[0065] The morphology and size of nanoparticles were determined
using transmission electron micrograph (TEM) and dynamic light
scattering (DLS). TEM of MnO nanoparticles in chloroform and
aqueous M-LMNs was performed by pipetting 10 .mu.l of diluted stock
solution (0.25 mg/ml) onto a carbon-coated copper grid. The MnO
grid was allowed to air-dry for one hour before visualization and
the M-LMNs grid was allowed to air-dry overnight. Once dry the
M-LMNs grid was then negatively stained using a 1% uranyl acetate
solution. The sample was visualized with a JEOL 1200 EX
transmission electron microscope at 80 kV. DLS of M-LMNs in aqueous
solution was performed using a DynaPro DLS plate reader. To prepare
DLS samples, the M-LMNs stock solution was diluted to a
concentration of 0.25 mg/ml and sonicated for 30 minutes to prevent
aggregation. Zeta potential was determined using a
MicroTracZetaTrac instrument. To prepare the samples, the M-LMNs
stock solution was diluted to 0.25 mg/ml and sonicated for 30
minutes to prevent aggregation.
[0066] TEM images of oleate-MnO showed mostly spherical
nanoparticles with a size of 10-30 nm. DLS analysis showed the
hydrodynamic radius for the M-LMNs to be about 100 nm, which was
confirmed by TEM images of M-LMNs where several electron-dense MnO
nanoparticles can be seen clustered within an M-LMN particle. The
surface charge of these micelles was determined by measuring their
zeta potential. M-LMNs showed a net positive zeta potential of +37
mV, most likely due to the cationic DC-cholesterol and DOPE with
its primary amine head groups. Inductively-coupled plasma-mass
spectrometry was used to determine the concentration of Mn
encapsulated in the M-LMNs. The MnO loading efficiency was
determined to be about 10%.
Example 2
Cellular Uptake, Cytotoxicity and in vivo Biodistribution of
M-LMNs
[0067] To examine the cellular uptake of M-LMNs, the particles were
labeled with the fluorescent lipid,
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-lissamine
rhodamine B sulfonyl. More specifically, fluorescent M-LMNs
(FM-LMNs) were prepared as previously described with some
modifications. The fluorescent-labeled lipid
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-lissamine
rhodamine B sulfonyl (0.2 mg) was added to the initial lipid
mixture in chloroform during micelle preparation. For the uptake
experiments, cells were seeded 24 hours prior to transfection into
an 8-well chamber slide at a density of 80,000 cells per well in
300 .mu.l of complete medium (DMEM containing 10% FBS, 2 mM
L-glutamate and 1% penicillin/streptomycin). At the time of FM-LMNs
addition, the medium in each well was replaced with 250 .mu.l of
fresh DMEM with no FBS. Various amounts of FM-LMNs, diluted in 50
.mu.l DMEM with no FBS, were added to each well. After 4 hours of
incubation, the cells were washed with PBS and fixed to the slide
using a 10% neutral buffered formalin solution. Nuclei of the cells
were stained using DAPI. The distribution of FM-LMNs inside the
cells was imaged with the multiphoton Olympus BX61W1 confocal
microscope. Human embryonic kidney HEK293 cells were incubated with
labeled M-LMNs for 4 hours and the dye was visualized by confocal
microscopy (FIG. 2A). M-LMNs were seen in the cytoplasm surrounding
the nuclei of the cells.
[0068] To determine the cytotoxicity of these micelles, cell
viability was measured using the PrShinaesto Blue assay. More
specifically, in vitro cytotoxicity was evaluated in HEK293 cells
and LLC1 cells using the PrestoBlue.RTM. assay (Roche) according to
the manufacturer's specifications. Cells were seeded 24 hours prior
to transfection into a 96-well plate in 100 .mu.l of complete
medium (DMEM containing 10% FBS, 2 mM L-glutamate and 1%
penicillin/streptomycin). At the time of nanoparticle addition, the
medium in each well was replaced with 50 .mu.l of fresh complete
DMEM. Various concentrations of the nanoparticles were diluted in
50 .mu.l DMEM with no FBS and added to the well in triplicate. The
cells were cultured in an incubator at 37.degree. C. under 5%
CO.sub.2 and viability was determined after 72 hours. Cells without
nanoparticles were used as a control with viability taken as 100%.
FIG. 2B shows that M-LMNs demonstrated no apparent toxicity when
incubated with the HEK cells at any of the concentrations
tested.
[0069] To determine the particles' biodistribution in vivo, M-LMNs
were labeled with Cy5.5, a near-infrared imaging dye. The resulting
particles were administered intravenously (IV) or intranasally (IN)
to groups of C57BL/6 mice. Control mice were administered PBS (IN).
Twenty-four, 48 and 96 hours after treatment, lung, liver, kidney,
and spleen were excised and examined for Cy5.5 using a Xenogen IVIS
imager and quantified. Twenty-four hours after IV administration,
the Cy5.5-M-LMNs were found mostly distributed in the liver and
also in the kidney and spleen, but not in the lungs of mice (FIG.
2C). In sharp contrast, intranasally administered Cy5.5-M-LMNs were
found preferentially (approximately 85% of total) accumulated in
the lungs for up to 48 hours (FIG. 2C), demonstrating that M-LMNs
have the potential to be used as theranostics for lung disease.
Example 3
Gene Delivery Potential of M-LMNs
[0070] In order for the micelles to act as a gene delivery vehicle,
they must be able to form a stable complex with nucleic acids
during transport and entry to release the DNA within the cells. The
capability of these cationic lipid micelles to form complexes with
DNA was evaluated using a gel-retardation assay. More specifically,
M-LMN complexes with different ratios of micelle:DNA were tested.
M-LMNs were diluted with PBS to a final concentration of 2
.mu.g/.mu.l and aliquots of M-LMNs and plasmid DNA solution were
diluted separately with an appropriate volume of PBS. The plasmid
DNA solution was then added slowly to the M-LMNs solution and
vortexed for 30 minutes. The M-LMN:DNA complexes were mixed with
loading buffer and loaded into individual wells of a 0.9% agarose
gel containing ethidium bromide. Gels were electrophoresed at room
temperature in Tris/Borate/EDTA buffer at 120V for 20 minutes. DNA
bands were visualized using a ChemiDoc TM XRS imaging system.
[0071] DNA that was bound to the micelles remained in the wells,
while unbound DNA migrated down the gel. It was observed that
M-LMNs were able to completely retard migration of the DNA at
weight ratios as low as 5:1 (M-LMNs:DNA) (FIG. 3). During DNA
delivery it is critical to protect the DNA from degradation by
nucleases. The absence of ethidium bromide staining in even the
wells that contain the M-LMNs:DNA complexes suggest that the M-LMNs
are able to fully protect the DNA from the ethidium bromide at
weight ratios as low as 5:1 (M-LMNs:DNA).
[0072] To further prove that the DNA was protected from nucleases
once complexed with the M-LMNs, M-LMNs:ptd-Tomato DNA complexes
were exposed to DNase-I (FIG. 3B). More specifically, M-LMN
complexes with different micelle:DNA ratios were tested. M-LMNs
complexed with 0.5 .mu.g plasmid DNA (pCMV-td-Tomato, Invitrogen)
at M-LMN:DNA (wt/wt) 5:1 or 2:1 or 0.5 .mu.g plasmid DNA alone were
incubated with 0.5 U DNase I (Roche) for 20 minutes at 37.degree.
C. To inactivate the DNase I, the solutions were then incubated at
75.degree. C. for 10 minutes. The samples were then mixed with
loading buffer and loaded into individual wells of a 0.8% agarose
gel containing ethidium bromide. Gels were electrophoresed at room
temperature in Tris/borate/EDTA buffer at 100 V for 40 minutes. DNA
bands were visualized using a ChemiDoc TM XRS imaging system.
[0073] The presence of partial DNA bands in the lane and ethidium
bromide staining in the well of the 2:1 (M-LMNs:DNA wt/wt) complex
shown in FIG. 3 suggest that the DNA was only partially complexed
to the micelles at this ratio. The DNA that was not fully
encapsulated within the micelles was completely degraded, as can be
seen from the absence of any DNA bands in this lane. However, the
DNA that was fully enclosed in the micelles was protected from
DNase I degradation, as can be seen from the ethidium bromide
staining present in the well of this lane. At a ratio of 5:1
(M-LMNs: DNA wt/wt) the DNA was fully protected from both DNase I
degradation and ethidium bromide staining, as judged by the absence
of any bands in the lanes or ethidium bromide staining in these
wells. These results suggest that M-LMNs, at a (M-LMNs: DNA wt/wt)
ratio of 5:1 or higher are able to completely protect the complexed
DNA from nuclease degradation.
[0074] The ability of M-LMNs to transduce DNA into cultured cells
and achieve protein expression was also determined by using a
plasmid DNA encoding red-fluorescent protein (RFP) as a reporter.
More specifically, cells were seeded 24 hours prior to transfection
into a 96-well plate at a density of 10,000 cells per well in 100
.mu.l of complete medium (DMEM containing 10% FBS, 2 mM L-glutamate
and 1% penicillin/streptomycin). At the time of transfection, the
medium in each well was replaced with 100 .mu.l of fresh DMEM with
no FBS. An amount of M-LMNs equivalent to the desired weight ratio
needed for use with 0.2 .mu.g of DNA plasmid expressing
red-fluorescent protein (pCMV-td-Tomato, Invitrogen) was added to
each well. Four hours after addition of M-LMNs, 50 .mu.l of DMEM
containing 40% FBS was added to each well and the plate was
incubated for a total of 96 hrs. All images were made with an
Olympus IX71 microscope equipped with an EXFO X-Cite Series 120
fluorescence excitation light source (.lamda.ex=554 nm,
.lamda.em=581 nm) and a DP-70 high-resolution digital camera.
Images were taken at 24, 48, and 96 hours post-transfection.
[0075] FIG. 4 shows the results of these experiments wherein cells
were incubated with various ratios of micelle:pDNA in M-LMN
complexes (FIG. 4A and 4B) and for various times (FIG. 4C and 4D).
The fluorescent images in FIGS. 4A and 4B show that M-LMNs at
micelle:pDNA weight ratios of 5:1 or 10:1 readily transfected
HEK293 cells. Transfection was less efficient at a ratio of 15:1.
Expression of RFP was seen as early as 24 hours and was maximal at
96 hours (FIG. 4C and 4D). Also, M-LMN:ptd-Tomato DNA (5:1) induced
protein expression levels similar to lipofectamine-transfected DNA
(data not shown). These results indicate that M-LMNs may be a
useful tool for the delivery of DNA into mammalian cells.
Example 4
M-LMNs provide MRI Capability in vitro and ex vivo
[0076] In addition to administering nucleic acids and small
molecules to target sites, this cationic lipid nanoparticle system
was also designed to act as a T1 MRI contrast agent to allow
monitoring of the effects of gene or drug delivery, It was
recognized that phospholipid-encapsulated oleic acid coated
nanoparticles are strongly protected from the outside aqueous
environment by a tight hydrophobic layer and that this may prevent
water protons from contacting the manganese nanoparticle surface
and could therefore lead to a lowering of the relaxivity [H. Duan
et al., The Journal of Physical Chemistry Letters 2008, 8127].
However, the DOPE component of the MLNs phospholipid micelle has
two unsaturated fatty acid tails, which serve to increase the
fluidity of the phospholipid micellar membrane. Since T1 contrast
agents need to have direct interaction with the surrounding water
protons to affect their relaxation times [Z. Zhen & J. Xie,
Theranostics 2012, 2, 45], this increased fluidity could allow for
more interaction of the manganese oxide nanoparticles and water
protons.
[0077] To determine whether M-LMNs were able to act as an effective
T1 MRI contrast agent, their relaxation properties were analyzed by
MR phantom imaging. Phantom MRI was performed as follows: Various
dilutions of M-LMNs and DM-LMNs were diluted in deionized water and
the concentration of manganese in the micelles was determined by
ICP-MS. Two hundred .mu.l aliquots of the various micelle solutions
were added to a 96-well plate in duplicate and MR images were
obtained using an Agilent ASR 310 7 Tesla MRI high-field scanner.
Fast Spin-Echo Multi-Slice (FSEMS) experiments were performed in
imaging mode to determine the measure of T1 values. Nonlinear
least-square fitting was performed using the MATLAB program
(Mathworks, Inc.) on a pixel-by-pixel basis. A region of interest
was drawn for each well, where the mean value was used to determine
the longitudinal molar relaxivity r1. The image was recorded with
Vnmrj 3.0. FIG. 5 shows the visual (A) and quantitative (B) T1 MRI
contrast provided by M-LMNs for various Mn concentrations. The R1
relaxivity of M-LMNs (1.17 mM-1s-1) was larger than the values
reported for MnO-SiO2-PEG/NH.sub.2 nanoparticles (0.47 mM-1s-1) [T.
D. Schladt et al, Journal of Materials Chemistry 2012, 9253] and
was comparable to the values reported for
PEG-phospholipid-encapsulated HMONs (1.417 mM-1s-1) [J. Shin et
al., Angew Chem Int Ed Engl 2009, 48, 321].
[0078] Since M-LMNs preferentially accumulate in the lungs of mice
after intranasal administration, whether M-LMNs would enhance the
T1 MRI contrast of the lungs was investigated. More specifically,
C57B1/6 mice (n=2) were treated with one intranasal instillation of
M-LMNs (50 .mu.l of a 0.7 mM Mn solution). Control mice (n=4) were
given PBS. After one hour, the mice were euthanized; the lungs were
collected and placed into a medical cassette to be viewed. MR
images were obtained using an Agilent ASR 310 7 Tesla MRI
high-field scanner. Gradient Echo Multi-Slice (GEMS) experiments
(flip angle=45.degree.; TR=175) were performed in imaging mode.
Nonlinear least-square fitting was performed using the MATLAB
program (Mathworks, Inc) on a pixel-by-pixel basis. A region of
interest was drawn around each lung and the mean value of the
signal intensity was determined in this area. The image was
recorded with Vnmrj 3.0. FIG. 5 shows the visual (C) and
quantitative (D) T1 MRI contrast provided by M-LMNs in mouse lungs.
The calculated mean signal intensity for M-LMN-injected lungs was
more than 2.5 times higher than that of the PBS-injected lungs.
These results demonstrate that in addition to administering a
therapeutic agent, the M-LMNs could potentially act as a T1 MRI
contrast agent to enhance detection and provide a more accurate
diagnosis or post-therapy evaluation.
Example 5
Cellular Uptake, Cytotoxicity and Biodistribution of Doxorubicin
(DOX)-Loaded LMNs (D-LMNs)
[0079] To determine the potential of LMNs to deliver small
molecular drugs, MnO in the hydrophobic core was replaced with the
chemotherapeutic drug DOX. More specifically, doxorubicin
hydrochloride (DOX) along with 4 molar equivalents of triethylamine
was added to chloroform and the mixture was sonicated for 30
minutes to dissolve the DOX. Phospholipid micelles encapsulating
DOX (referred to as D-LMNs) or DOX and MnO (referred to as DM-LMNs)
were prepared as previously described with some modifications [R.
Kumar et al., Theranostics 2012, 714-722; Y. Namiki et al., Nature
Nanotechnology 2009, 4, 598]. The 3 mg of MnO was replaced by 3 mg
of DOX in D-LMNs, and in DM-LMNs, the 3 mg of MnO was replaced by
1.5 mg of DOX and 1.5 mg of MnO. D-LMNs were found to be spherical,
as judged by TEM (FIG. 6A) with a hydrodynamic radius of about 100
nm and a positive zeta potential.
[0080] To evaluate the potential of these nanoparticles for
therapeutic delivery, in vitro cellular uptake of D-LMNs was
evaluated. Cellular uptake experiments using D-LMNs and DM-LMNs
were performed in the same manner as the FM-LMNs uptake studies.
Cells were seeded 24 hours prior to transfection into an 8-well
chamber slide at a density of 80,000 cells per well in 300 .mu.l of
complete medium (DMEM containing 10% FBS, 2 mM L-glutamate and 1%
penicillin/streptomycin). At the time of FM-LMNs addition, the
medium in each well was replaced with 250 .mu.l of fresh DMEM
without FBS. Various amounts of FM-LMNs, diluted in 50 .mu.l DMEM
with no FBS, were added to each well. After 4 hours of incubation,
the cells were washed with PBS and fixed with 10% neutral buffered
formalin. Nuclei were stained with DAPI. The distribution of
FM-LMNs inside the cells was determined with a multiphoton Olympus
BX61W1 confocal microscope.
[0081] Fluorescence images of HEK293 cells incubated with D-LMNs
for 24 hours showed that most of the DOX was distributed in the
cytoplasm of the cell (FIG. 6B). This is in contrast to cells
incubated with free DOX, where the DOX is found in the nuclei
intercalated with DNA [R. Kumar et al., Theranostics 2012,
714-722]. These data suggest that the internalization mechanism of
the D-LMNs is different from that of free DOX. Similar results have
been reported before by other groups using micellar carriers to
deliver DOX to cells [X. Shuai et al., Journal of Controlled
Release: Official Journal of the Controlled Release Society 2004,
98, 415].
[0082] An in vitro DOX release study was also performed wherein
D-LMNs, M-LMNs, and free DOX were each dispersed in 1 ml PBS buffer
containing 1% Tween 20 (pH 7.3 or pH 5.1) and placed in a dialysis
membrane (MWCO of 12000-14000 Da), The bag was then immersed in a
tube containing 10 mL of the same PBS buffer (pH 7.4 or pH 5.1) and
incubated at 37.degree. C. At specific time intervals the DOX
content in the PBS was analyzed using the UV-VIS spectrophotometer
at 485 nm. Samples were all done in triplicate. M-LMNs were used as
a blank. The release of the encapsulated DOX from D-LMNs was
determined at pH 7.3, which is the physiological pH, and at pH 5.1,
which represents the acidic pH inside endosomes, lysosomes, and
solid tumors. One percent Tween 20 was used because it forms
hydrophobic pockets, which can stabilize the released DOX from the
D-LMNs, and helps to avoid aggregation of the hydrophobic DOX in
the aqueous environment.
[0083] The release profile of DOX from D-MLNs is shown in FIG. 6C.
DOX release occurred with an initial burst during the first 6 hours
with about 50% of the DOX releasing at pH 7.3 and about 40% of the
DOX releasing at pH 5.1. Subsequently, the release occurred more
slowly and steadily with more than 90% of the free DOX being
released into the solution at either pH after 96 hours. However,
even after 48 hours only 48% and 68% of the DOX had been released
from the D-LMNs at pH 7.3 and pH 5.1, respectively. These results
demonstrate that the DOX is sequestered within the micelles and
that the pH of the environment plays a role in DOX release from the
micelles. This moderate pH-dependent release may be due to the
protonation of the amine head group on DOPE in an acidic
environment, which could be causing destabilization of the micelle
and subsequent release of the contents [H. Farhood, N. Serbina
& L. Huang, Biochimica et Biophysica Acta 1995, 1235, 289].
[0084] To determine whether D-LMNs can deliver active free DOX,
LLC1 cells were incubated with D-LMNs containing various
concentrations of DOX, and cell viability after 72 hours was
determined. More specifically, D-LMNs, M-LMNs, and free DOX were
each dispersed in 1 ml PBS buffer containing 1% Tween 20 (pH 7.3 or
pH 5.1) and placed in a dialysis membrane (MWCO of 12000-14000 Da).
The bag was then immersed in a tube containing 10 mL of the same
PBS buffer (pH 7.4 or pH 5.1) and incubated at 37.degree. C. At
specific time intervals the DOX content in the PBS was analyzed
using the UV-VIS spectrophotometer at 485 nm. Samples were all done
in triplicate. M-LMNs were used as a blank. FIG. 6D shows the
results of this study. The D-LMNs are just as toxic to the cells as
free DOX when used at the same DOX concentrations (FIG. 6E), It is
also clear that these toxic effects are due solely to the DOX and
not the other components of the micelles, as M-LMNs alone exerted
no cytotoxic effects (FIG. 6D). These results demonstrate that
D-LMNs can deliver DOX as a payload to kill tumor cells.
[0085] To study the in vivo biodistribution and safety of D-LMNs,
C57B1/6 mice were treated with six rounds of intranasal
instillations of D-LMNs over a period of two weeks. More
specifically, C57B1/6 mice were treated with six rounds of
intranasal instillations of D-LMNs (50 .mu.l containing 0.532 mM
DOX solution) over a period of two weeks. Control mice were
administered PBS. The mice were then euthanized and the lung,
liver, kidney, spleen, and pancreas were collected. The
biodistribution of DOX in each organ was viewed using the Xenogen
IVIS-200 Optical In Vivo Imaging System. The lung, liver, and
kidney were then stored in OCT and frozen at -80.degree. C. These
organs were then sectioned, stained with hematoxylin and eosin, and
examined for changes in morphology. For biodistribution studies,
one round of D-LMNs (50 .mu.l containing 1.1 mM DOX solution) was
administered intranasally (IN) to C57BL/6 mice. Control mice were
administered PBS (IN). Twenty-four and 48 hours after the
administration, lung, liver, and kidney were excised. The lung,
liver, and kidney were then stored in OCT and frozen at -80.degree.
C. These organs were then sectioned and viewed using fluorescence
microscopy to determine DOX uptake.
[0086] From FIG. 6F it can be seen that, when administered
intranasally, the D-LMNs preferentially accumulate and release DOX
in the lungs. The relatively low levels of DOX in the other organs
suggest that D-LMN nanoparticles outside the lung are efficiently
cleared from the body. All of these results together demonstrate
the potential of D-LMNs for administering chemotherapeutic agents
for the treatment of lung cancer.
[0087] To evaluate potential toxicity of the D-LMNs in vivo organ
sections were stained with hematoxylin/eosin (H&E) and examined
by light microscopy. It is well known that high levels of free DOX
are highly toxic to tissues and can cause ulcerations and necrosis.
Despite relatively high levels of DOX accumulation in the lungs,
liver, and kidneys, which was confirmed by biodistribution studies
(FIG. 6F), no morphological or histological alterations in the
organs were observed (data not shown). The reduction of systemic
toxicity can most likely be attributed to the DOX being sequestered
within the cationic lipid nanoparticles and not being released in
the bloodstream. These studies demonstrate that D-LMNs are able to
minimize the chemotherapeutic side effects of DOX on susceptible
organs.
Example 6
Multifunctional LMNs for Simultaneous Delivery of MnO, pDNA and
DOX
[0088] To evaluate the potential of LMNs to deliver functional MnO
as a T1 contrast agent, DOX for chemotherapy and plasmid DNA for
gene therapy, we synthesized a multifunctional LMN incorporating a
mixture of hydrophobic DOX and MnO in the core and the
negatively-charged pDNA on the positively-charged surface. The
resulting particles, which are referred to as DM-LMNs, had a
positive zeta potential and spherical morphology with a diameter
similar to M-LMNs (200 nm). To examine whether DM-LMNs were also
capable of providing efficient T1 MRI contrast, the DM-LMNs were
analyzed using the same MR phantom imaging as M-LMNs. At a
concentration of 1 mM, DM-LMNs were able to provide a T1 relaxivity
that was only slightly less than that of M-LMNs (data not shown).
These results suggest that these micelles can provide effective T1
MRI contrast.
[0089] HEK293 cells were incubated with DM-LMNs for 24 hours and
DOX uptake was determined by analysis of confocal fluorescence
microscope images. In vitro transfections using DM-LMNs were
carried out in the same manner as transfections using M-LMNs,
except that the simultaneous GFP/DOX transfection was done using an
8-well chamber slide instead of a 96-well plate for imaging
purposes. HEK293 cells were plated with a density of 80,000 cells
per well in 300 .mu.l of complete medium (DMEM containing 10% FBS,
2 mM L-glutamate and 1% penicillin/streptomycin). After 48 hours,
the cells were fixed onto the slide using 10% neutral buffered
formalin and viewed for GFP and DOX using fluorescence microscopy.
Similar to D-LMNs, DOX was seen mostly in the cytoplasm of the
cells (FIG. 7A). Treatment of LLC1 cells with DM-LMNs for 72 hours
showed cytotoxicity comparable to that seen with LLC1 cells
incubated with free DOX. With DOX concentrations of 1 .mu.M or
higher, over 50% of the cells were killed (FIG. 7B). These results
show that DM-LMNs can deliver DOX as efficiently as D-LMNs while
still retaining MRI contrast ability.
[0090] It was then determined whether DM-LMNs could deliver nucleic
acids as efficiently as M-LMNs. HEK293 cells were transfected with
DM-LMNs at the same DM-LMNs:ptd-Tomato DNA weight ratios as M-LMNs.
The results (FIGS. 7C and 7D) show that DM-LMNs were capable of
administering ptd-Tomato DNA to HEK293 cells with slightly less
efficiency than M-LMNs. This can most likely be attributed to the
loss of cells due to DOX-induced cytotoxicity. To image the
simultaneous delivery of DNA and DOX, HEK293 cells were incubated
with DM-LMNs complexed with DNA encoding enhanced green fluorescent
protein (EGFP) for 48 hours. HEK293 cells can be seen with DOX
located throughout the cytoplasm, similar to D-LMNs, and EGFP
expression throughout the cytoplasm (FIG. 7E). These data show that
DM-LMNs can simultaneously deliver nucleic acids and
chemotherapeutic agents into cells.
[0091] To determine if DM-LMNs are capable of simultaneously
administering DNA and DOX in vivo, nanoparticles complexed with
EGFP-DNA were administered intranasally to mice. More specifically,
C57BL/6 mice were administered 125 .mu.g of M-LMNs complexed with
25 .mu.g EGFP-DNA in 50 .mu.l PBS (n=2) or 125 .mu.g of DM-LMNs
complexed with 25 .mu.g GFP-DNA (with 0.25 mM DOX in 50 .mu.l)
(n=2) intranasally. Seventy-two and 96 hours after the
administration, lung, liver, and kidney were excised. The lung,
liver, and kidney were then stored in OCT and frozen at -80.degree.
C. The organs were sectioned and stained for GFP using anti-GFP
antibody, then viewed for GFP expression and DOX uptake using
fluorescent microscopy.
[0092] At 72 and 96 hours after administration, mice were
euthanized and the lungs, liver, and kidneys were collected in OCT
and frozen at -80.degree. C. Frozen sections were immunostained for
EGFP and analyzed for GFP expression and DOX uptake using
fluorescence microscopy. EGFP and DOX expression could be seen in
the lungs at both 72 and 96 hours (FIG. 7F). These results suggest
that, regardless of the payload. LMNs are able to preferentially
accumulate in the lungs when delivered intranasally and that these
nanoparticles are capable of the simultaneous delivery of DNA and
DOX in vivo. Taken together, these experimental results show that
LMNs provide a simple and efficient theranostic micellar
formulation that can be used as a multifunctional vehicle for
imaging and therapy of cancer in vitro and in vivo.
* * * * *